Project Summary/Abstract Neurological dysfunction is a significant complication of HIV disease. Despite the success of HAART in suppressing plasma HIV RNA, most antiretroviral drugs have poor CNS penetration and a reservoir of virus persists in brain. This viral reservoir impedes the goal of eradicating HIV, emphasizing the need to understand mechanisms of HIV neurotropism and neurovirulence. During the current funding period, we showed that HIV Envs with reduced dependence on CD4 and CCR5 are more frequent in brain compared to lymphoid tissues, suggesting that viral adaptation for replication in the CNS selects for variants with an enhanced capacity to enter cells expressing low levels of receptor. We identified a specific variant in the CD4 binding site of HIV gp120 (i.e., N283) that is more frequent in Envs from brain than lymphoid tissue (p<0.01), and in viruses from HIV-associated dementia (HAD) than non-HAD patients (p<0.001). The N283 variant increases gp120 affinity for CD4 by decreasing the gp120-CD4 dissociation rate, enhancing the capacity of Envs to enter cells expressing low levels of CD4 and enhancing viral replication in macrophages/microglia. Although HAART has improved survival, HIV-associated neurocognitive impairment and HIV encephalitis (HIVE) are still prevalent. Compared to AIDS patients of the pre-HAART era, patients of the HAART era have lower levels of HIV RNA and immune activation in brain. Accordingly, biological properties of HIV in the brain of pre- and post-HAART patients may differ substantially. Much remains to be learned about entry events critical for selection and adaptation of virus to the CNS, biological properties of HIV in brain in HAART compared to pre-HAART patients, the ability of HIV to modulate cellular reactions (e.g., immune activation and mitochondrial injury) involved in neuronal injury, and the association between the virus and differences in neurological disease in the pre- and post-HAART eras. Here we will systematically examine the virus and its biological properties that influence neurotropism and cellular processes that contribute to neuropathogenesis using autopsy samples from brain and other tissues from patients with HIV-associated neurological disease before and after the advent of HAART. Specifically, we will: 1) determine the genetic and biological properties of HIV Envs in virus in brain from patients on HAART with neurocognitive impairment and whether they differ from those of pre- HAART patients, and investigate the role of cellular reactions (e.g., immune activation and mitochondrial injury) in this process;2) investigate the role of virion fusion kinetics in macrophages/microglia tropism, and characterize the properties of brain-derived Envs that influence these properties through mechanisms other than Env-receptor affinity;and 3) determine whether HIV in brain shares signature sequences and biological properties with viruses in macrophages in GALT and/or lung in patients from the pre- and/or post-HAART eras. The studies will provide a better understanding of mechanisms of HIV neuropathogenesis, and insights that may facilitate development of therapies to prevent CNS infection and neurologic injury in HIV-infected patients.